An optimized isolation and labeling platform for accurate microRNA expression profiling
An optimized isolation and labeling platform for accurate microRNA expression profiling
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DOI：
10.1261/rna.2610405
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发表时间：
2005-09-01
期刊：
影响因子：
4.5
通讯作者：
Labourier, E
中科院分区：
文献类型：
--
作者：
Shingara, J;Keiger, K;Labourier, E
MicroRNAs(MiRNAs)是一种小的、非编码的RNA，调节植物和动物的基因表达。MiRNA基因参与了多种重要的生物学过程，包括发育、分化、细胞凋亡、脂肪代谢、病毒感染和癌症。与编码蛋白质的信使RNA类似，miRNA的表达在组织和发育状态之间也不同。为了更好地了解miRNA在组织和细胞中的全球表达，我们开发了分离、标记和阵列程序来测量所有已知的人类成熟miRNAs的相对丰度。该方法依赖于快速分离小于40个核苷酸(NT)的RNA物种，用胺修饰的核苷酸直接和同源标记成熟的miRNAs，并与反义DNA寡核苷酸探针杂交。一项全面的性能研究表明，该miRNA微阵列系统可以从1微克总RNA中检测出亚毫微摩尔量的单个miRNA，独立重复之间的相关性为98%。该系统已经被应用于比较26个不同的正常人体组织中的全球miRNA表达谱。这一综合分析确定了在一个或几个相关组织中优先表达的miRNAs，并揭示了人类成人组织具有独特的miRNA图谱。这意味着miRNAs是组织发育和分化的重要组成部分。综上所述，这些结果强调了微阵列在正常和疾病状态下对miRNA表达进行灵敏和高通量分析的巨大潜力。
MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in both plants and animals. miRNA genes have been implicated in a variety of important biological processes, including development, differentiation, apoptosis, fat metabolism, viral infection, and cancer. Similar to protein-coding messenger RNAs, miRNA expression varies between tissues and developmental states. To acquire a better understanding of global miRNA expression in tissues and cells, we have developed isolation, labeling, and array procedures to measure the relative abundance of all of the known human mature miRNAs. The method relies on rapid isolation of RNA species smaller than similar to 40 nucleotides (nt), direct and homogenous enzymatic labeling of the mature miRNAs with amine modified ribonucleotides, and hybridization to antisense DNA oligonucleotide probes. A thorough performance study showed that this miRNA microarray system can detect subfemtomole amounts of individual miRNAs from < 1 mu g of total RNA, with 98% correlation between independent replicates. The system has been applied to compare the global miRNA expression profiles in 26 different normal human tissues. This comprehensive analysis identified miRNAs that are preferentially expressed in one or a few related tissues and revealed that human adult tissues have unique miRNA profiles. This implicates miRNAs as important components of tissue development and differentiation. Taken together, these results emphasize the immense potential of microarrays for sensitive and high-throughput analysis of miRNA expression in normal and disease states.